Subtelomeres are likely the most structurally complex, variable, and dynamic regions of the human genome. Subtelomeric exchanges have distributed sequences to multiple chromosome ends and resulted in extensive variation in subtelomeric content. Information is still fragmentary, but subtelomeres contain genes, making interindividual variation a potential source of phenotypic diversity. Subtelomeric homology may also mediate deleterious rearrangements to cause disease and/or play a role in the ALT pathway, enabling cancer cells to proliferate indefinitely without telomerase. Our long-term goal is to understand how the unusual characteristics of subtelomeres impact phenotypic diversity, speciation, and disease. Our three aims are to characterize the (1) structure, variability, and evolution, (2) gene content and function, and (3) mitotic and meiotic dynamics of subtelomeres. The project builds on our expertise in FISH, genome analysis, and chromosome sorting and complements efforts being made elsewhere to sequence a representative allele of each subtelomere. Aim 1 has four sub-aims. (1a) By using a combined computational and experimental approach, we will determine the boundaries of multicopy blocks and assess polymorphism in their number and location. (1b) We will test the hypothesis that subtelomeric duplications spread only recently, by using FISH to locate various blocks in other primates and Old and New World monkeys. (1c) We will isolate and map clones from "orthologous" subtelomeric regions in several species for sequencing by Eric Green's group, in order to identify sequences that may have been lost as humans diverged from other primates. (1d) And, as a proof of principle, we will perform targeted cloning and sequencing of a variant 7p allele found predominantly in Africans. In Aim 2, we will analyze each of ~20 subtelomeric gene families for potential function and expression and assess variation in gene sequence and number among humans and primates. In Aim 3, we will analyze the involvement of subtelomeric regions in recombination events. (3a) We will conduct CO-FISH assays to determine if sister chromatid exchanges (SCEs) within subtelomeric zones account for the unusually high SCE frequencies attributed to terminal bands of chromosomes. (3b) We will develop and deploy an assay for inter-chromosomal mitotic exchange between subtelomeres in normal, DNA repair-deficient, and ALT cells. (3c) And, we will determine how meiotic mispairing of chromosome ends relates to subtelomeric sequence homology. Our genomic and functional analyses of these complex regions of the human genome should provide new insights into how sharp the double-edged sword of subtelomere dynamics is in mediating adaptive change and deleterious rearrangements.